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Abstract:

The present invention relates to the use of butaclamol and pharmaceutical
compositions thereof for treating subjects with amyotrophic lateral
sclerosis (ALS) and other neurodegenerative diseases.

Claims:

1. A method comprising steps of: administering to a subject suffering
from or susceptible to amyotrophic lateral sclerosis an effective amount
of butaclamol, such that the severity or incidence of one or more
symptoms of ALS is reduced, or its onset is delayed.

2. The method of claim 1, wherein the butaclamol is administered in the
form of a salt.

3. A method comprising steps of: administering to a subject suffering
from or susceptible to abnormal protein aggregation an amount of
butaclamol sufficient to reduce or delay such abnormal protein
aggregation.

[0004] The present invention encompasses the recognition that there exists
a need for methods for treating patients with amyotrophic lateral
sclerosis (ALS) or other neurodegenerative diseases characterized by the
presence of aberrant protein aggregates.

[0005] The present invention relates to the use of butaclamol to treat
neurodegenerative diseases characterized by abberrant protein aggregates.
Among other things, the present invention provides methods of treating
amyotrophic lateral sclerosis (ALS) with butaclamol. Without wishing to
be bound by any particular theory, butaclamol may be useful in the
treatment of (ALS) or other neurodegenerative diseases where abnormal
protein aggregation has been implicated, as it may prevent the
aggregation of protein in a cell or limit the toxicity of such
aggregates.

[0006] In one aspect, the invention provides methods of treating a subject
suffering from or susceptible to a neurodegenerative disease, disorder or
condition (e.g., ALS) with butaclamol. In certain embodiments, the
subject is an adult human.

[0007] In some embodiments, the present invention provides methods of
inhibiting or reversing abnormal protein aggregation (e.g., SOD1 protein
aggregates) using butaclamol. Inhibiting or reversing abnormal protein
aggregation may occur in vivo (e.g., in a subject as described herein) or
in vitro (e.g., in a cell).

[0008] In some embodiments, the invention provides methods of protecting
cells from the cytotoxic effects of aggregated protein (e.g., SOD1) using
butaclamol. Protection of cells may occur in vivo (e.g., in a subject as
described herein) or in vitro (e.g., in a cell).

[0009] In some embodiments, the invention provides methods of modulating
proteasome activity in vivo (e.g., in a subject as described herein) or
in vitro (e.g., in a cell) using an inventive compound. In certain
embodiments, the cells are mammalian cells.

[0010] All publications and patent documents cited in this application are
incorporated herein by reference in their entirety.

DEFINITIONS

[0011] Animal: As used herein, the term "animal" refers to any member of
the animal kingdom. In some embodiments, "animal" refers to humans, at
any stage of development. In some embodiments, "animal" refers to
non-human animals, at any stage of development. In certain embodiments,
the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a
rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a
pig). In some embodiments, animals include, but are not limited to,
mammals, birds, reptiles, amphibians, fish, and/or worms. In some
embodiments, an animal may be a transgenic animal, a
genetically-engineered animal, and/or a clone.

[0012] Approximately: As used herein, the terms "approximately" or "about"
in reference to a number are generally taken to include numbers that fall
within a range of 5%, 10%, 15%, or 20% in either direction (greater than
or less than) of the number unless otherwise stated or otherwise evident
from the context (except where such number would be less than 0% or
exceed 100% of a possible value). In some embodiments, use of the term
"about" in reference to dosages means±5 mg/kg/day.

[0013] Characteristic portion: As used herein, the phrase a
"characteristic portion" of a protein or polypeptide is one that contains
a continuous stretch of amino acids, or a collection of continuous
stretches of amino acids, that together are characteristic of a protein
or polypeptide. Each such continuous stretch generally will contain at
least two amino acids. Furthermore, those of ordinary skill in the art
will appreciate that typically at least 5, 10, 15, or more amino acids
are required to be characteristic of a protein. In general, a
characteristic portion is one that, in addition to the sequence identity
specified above, shares at least one functional characteristic with the
relevant intact protein.

[0014] Characteristic sequence: A "characteristic sequence" is a sequence
that is found in all members of a family of polypeptides or nucleic
acids, and therefore can be used by those of ordinary skill in the art to
define members of the family.

[0015] Intraperitoneal: The phrases "intraperitoneal administration" and
"administered intraperitonealy" as used herein have their art-understood
meaning referring to administration of a compound or composition into the
peritoneum of a subject.

[0016] In vitro: As used herein, the term "in vitro" refers to events that
occur in an artificial environment, e.g., in a test tube or reaction
vessel, in cell culture, etc., rather than within an organism (e.g.,
animal, plant, and/or microbe).

[0017] In vivo: As used herein, the term "in vivo" refers to events that
occur within an organism (e.g., animal, plant, and/or microbe).

[0018] Oral: The phrases "oral administration" and "administered orally"
as used herein have their art-understood meaning referring to
administration by mouth of a compound or composition.

[0019] Parenteral: The phrases "parenteral administration" and
"administered parenterally" as used herein have their art-understood
meaning referring to modes of administration other than enteral and
topical administration, usually by injection, and include, without
limitation, intravenous, intramuscular, intraarterial, intrathecal,
intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,
transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,
subarachnoid, intraspinal, and intrasternal injection and infusion.

[0020] Patient: As used herein, the term "patient", "subject", or "test
subject" refers to any organism to which butaclamol is administered in
accordance with the present invention e.g., for experimental, diagnostic,
prophylactic, and/or therapeutic purposes. Typical subjects include
animals (e.g., mammals such as mice, rats, rabbits, non-human primates,
and humans; insects; worms; etc.). In some embodiments, a subject may be
suffering from, and/or susceptible to a disease, disorder, and/or
condition (e.g., a neurodegenerative disease, a disease, disorder or
condition associated with protein aggregation, ALS, etc.).

[0021] Pharmaceutically acceptable: The phrase "pharmaceutically
acceptable" is employed herein to refer to those compounds, materials,
compositions, and/or dosage forms which are, within the scope of sound
medical judgment, suitable for use in contact with the tissues of human
beings and animals without excessive toxicity, irritation, allergic
response, or other problem or complication, commensurate with a
reasonable benefit/risk ratio.

[0022] Prodrug: A general, a "prodrug", as that term is used herein and as
is understood in the art, is an entity that, when administered to an
organism, is metabolized in the body to deliver a therapeutic agent of
interest. Various forms of "prodrugs" are known in the art. For examples
of such prodrug derivatives, see: [0023] a) Design of Prodrugs, edited by
H. Bundgaard, (Elsevier, 1985) and Methods in Enzymology, 42:309-396,
edited by K. Widder, et al. (Academic Press, 1985); [0024] b) A Textbook
of Drug Design and Development, edited by Krogsgaard-Larsen; [0025] c)
Bundgaard, Chapter 5 "Design and Application of Prodrugs", by H.
Bundgaard, p. 113-191 (1991); [0026] d) Bundgaard, Advanced Drug Delivery
Reviews, 8:1-38 (1992); [0027] e) Bundgaard, et al., Journal of
Pharmaceutical Sciences, 77:285 (1988); and [0028] f) Kakeya, et al.,
Chem. Pharm. Bull., 32:692 (1984).

[0029] The methods and structures described herein relating to butaclamol
compounds also apply to pharmaceutically acceptable salts thereof.

[0030] Protein: As used herein, the term "protein" refers to a polypeptide
(i.e., a string of at least two amino acids linked to one another by
peptide bonds). In some embodiments, proteins include only
naturally-occurring amino acids. In some embodiments, proteins include
one or more non-naturally-occurring amino acids (e.g., moieties that form
one or more peptide bonds with adjacent amino acids). In some
embodiments, one or more residues in a protein chain contains a
non-amino-acid moiety (e.g., a glycan, etc). In some embodiments, a
protein includes more than one polypeptide chain, for example linked by
one or more disulfide bonds or associated by other means. In some
embodiments, proteins contain L-amino acids, D-amino acids, or both; in
some embodiments, proteins contain one or more amino acid modifications
or analogs known in the art. Useful modifications include, e.g., terminal
acetylation, amidation, methylation, etc. The term "peptide" is generally
used to refer to a polypeptide having a length of less than about 100
amino acids, less than about 50 amino acids, less than 20 amino acids, or
less than 10 amino acids. In some embodiments, proteins are antibodies,
antibody fragments, biologically active portions thereof, and/or
characteristic portions thereof.

[0031] Stereochemically isomeric forms: The phrase "stereochemically
isomeric forms", as used herein, refers to different compounds made up of
the same atoms bonded by the same sequence of bonds but having different
three-dimensional structures which are not interchangeable. In some
embodiments of the invention, chemical compositions may be provided as
pure preparations of individual stereochemically isomeric forms of a
compound; in some embodiments, chemical compositions may be provided that
are or include mixtures of two or more stereochemically isomeric forms of
the compound. In certain embodiments, such mixtures contain equal amounts
of different stereochemically isomeric forms; in certain embodiments,
such mixtures contain different amounts of at least two different
stereochemically isomeric forms. In some embodiments, a chemical
composition may contain all diastereomers and/or enantiomers of the
compound. In some embodiments, a chemical composition may contain less
than all diastereomers and/or enantiomers of a compound. Unless otherwise
indicated, the present invention encompasses all stereochemically
isomeric forms of relevant compounds, whether in pure form or in
admixture with one another. If a particular enantiomer of a compound of
the present invention is desired, it may be prepared, for example, by
asymmetric synthesis, or by derivation with a chiral auxiliary, where the
resulting diastereomeric mixture is separated and the auxiliary group
cleaved to provide the pure desired enantiomers. Alternatively, where the
molecule contains a basic functional group, such as amino, diastereomeric
salts are formed with an appropriate optically-active acid, and resolved,
for example, by fractional crystallization.

[0032] Substantially: As used herein, the term "substantially" refers to
the qualitative condition of exhibiting total or near-total extent or
degree of a characteristic or property of interest. One of ordinary skill
in the biological arts will understand that biological and chemical
phenomena rarely, if ever, go to completion and/or proceed to
completeness or achieve or avoid an absolute result. The term
"substantially" is therefore used herein to capture the potential lack of
completeness inherent in many biological and/or chemical phenomena.

[0033] Suffering from: An individual who is "suffering from" a disease,
disorder, and/or condition has been diagnosed with and/or displays one or
more symptoms of a disease, disorder, and/or condition.

[0034] Susceptible to: An individual who is "susceptible to" a disease,
disorder, and/or condition is one who has a higher risk of developing the
disease, disorder, and/or condition than does a member of the general
public. In some embodiments, an individual who is susceptible to a
disease, disorder and/or condition may not have been diagnosed with the
disease, disorder, and/or condition. In some embodiments, an individual
who is susceptible to a disease, disorder, and/or condition may exhibit
symptoms of the disease, disorder, and/or condition. In some embodiments,
an individual who is susceptible to a disease, disorder, and/or condition
may not exhibit symptoms of the disease, disorder, and/or condition. In
some embodiments, an individual who is susceptible to a disease,
disorder, and/or condition will develop the disease, disorder, and/or
condition. In some embodiments, an individual who is susceptible to a
disease, disorder, and/or condition will not develop the disease,
disorder, and/or condition.

[0035] Tautomeric forms: The phrase "tautomeric forms", as used herein, is
used to describe different isomeric forms of organic compounds that are
capable of facile interconversion. Tautomers may be characterized by the
formal migration of a hydrogen atom or proton, accompanied by a switch of
a single bond and adjacent double bond. In some embodiments, tautomers
may result from prototropic tautomerism (i.e., the relocation of a
proton). In some embodiments, tautomers may result from valence
tautomerism (i.e., the rapid reorganization of bonding electrons). All
such tautomeric forms are intended to be included within the scope of the
present invention. In some embodiments, tautomeric forms of a compound
exist in mobile equilibrium with each other, so that attempts to prepare
the separate substances results in the formation of a mixture. In some
embodiments, tautomeric forms of a compound are separable and isolatable
compounds. In some embodiments of the invention, chemical compositions
may be provided that are or include pure preparations of a single
tautomeric form of a compound. In some embodiments of the invention,
chemical compositions may be provided as mixtures of two or more
tautomeric forms of a compound. In certain embodiments, such mixtures
contain equal amounts of different tautomeric forms; in certain
embodiments, such mixtures contain different amounts of at least two
different tautomeric forms of a compound. In some embodiments of the
invention, chemical compositions may contain all tautomeric forms of a
compound. In some embodiments of the invention, chemical compositions may
contain less than all tautomeric forms of a compound. In some embodiments
of the invention, chemical compositions may contain one or more
tautomeric forms of a compound in amounts that vary over time as a result
of interconversion. Unless otherwise indicated, the present invention
encompasses all tautomeric forms of relevant compounds, whether in pure
form or in admixture with one another.

[0036] Therapeutic agent: As used herein, the phrase "therapeutic agent"
refers to any agent that, when administered to a subject, has a
therapeutic effect and/or elicits a desired biological and/or
pharmacological effect. In some embodiments, a therapeutic agent is any
substance that can be used to alleviate, ameliorate, relieve, inhibit,
prevent, delay onset of, reduce severity of, and/or reduce incidence of
one or more symptoms or features of a disease, disorder, and/or
condition.

[0037] Therapeutically effective amount: As used herein, the term
"therapeutically effective amount" means an amount of a substance (e.g.,
a therapeutic agent, composition, and/or formulation) that elicits a
desired biological response when administered as part of a therapeutic
regimen. In some embodiments, a therapeutically effective amount of a
substance is an amount that is sufficient, when administered to a subject
suffering from or susceptible to a disease, disorder, and/or condition,
to treat, diagnose, prevent, and/or delay the onset of the disease,
disorder, and/or condition. As will be appreciated by those of ordinary
skill in this art, the effective amount of a substance may vary depending
on such factors as the desired biological endpoint, the substance to be
delivered, the target cell or tissue, etc. For example, the effective
amount of compound in a formulation to treat a disease, disorder, and/or
condition is the amount that alleviates, ameliorates, relieves, inhibits,
prevents, delays onset of, reduces severity of and/or reduces incidence
of one or more symptoms or features of the disease, disorder, and/or
condition. In some embodiments, a therapeutically effective amount is
administered in a single dose; in some embodiments, multiple unit doses
are required to deliver a therapeutically effective amount.

[0038] Treat: As used herein, the term "treat," "treatment," or "treating"
refers to any method used to partially or completely alleviate,
ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity
of, and/or reduce incidence of one or more symptoms or features of a
disease, disorder, and/or condition. Treatment may be administered to a
subject who does not exhibit signs of a disease, disorder, and/or
condition. In some embodiments, treatment may be administered to a
subject who exhibits only early signs of the disease, disorder, and/or
condition, for example for the purpose of decreasing the risk of
developing pathology associated with the disease, disorder, and/or
condition.

[0039] Systemic: The phrases "systemic administration," "administered
systemically," "peripheral administration," and "administered
peripherally" as used herein have their art-understood meaning referring
to administration of a compound or composition such that it enters the
recipient's system.

[0040] For purposes of this invention, the chemical elements are
identified in accordance with the Periodic Table of the Elements, CAS
version, Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside
cover.

BRIEF DESCRIPTION OF THE DRAWING

[0041] FIG. 1. Proteasome inhibition is selectively toxic to PC12 cells
expressing mutant G93A SOD1. Cell survival was determined using the
viability stain calcein-AM. At 24 hr, treatment with 100 nM MG132 shows
no toxicity against any of the cell lines although aggregates are seen in
G85R SOD1 and G93A SOD1 cell lines (panel A). Treatment with 100 nM MG132
is selectively toxic to the G93A SOD1 cell line after 48 hours (panel B).
Washing of the cells at 24 hours to remove the compound does not reverse
toxicity to the G93A SOD1 cell line suggesting that an irreversible toxic
event, potentially related the aggregation of G93A SOD1, has been
triggered prior to wash out (panel C).

[0044] FIG. 4. Radicicol decreases mutant SOD1 aggregation induced by
proteasome inhibitor MG132. Fluorescence micrographs of PC12 cells
expressing YFP tagged G93A SOD1 (left) or G85R SOD1 (right) proteins. The
cells were treated with 200 nM MG132 to induce protein aggregation, or
co-treated with MG132 and radicicol for 24 h. Without radicicol
treatment, cells show large perinuclear aggregates. The aggregates are
reduced in radicicol-treated cells. While the behavior of the two cell
lines is generally similar, G85R SOD1 cells show `brighter` aggregates
and more contrast between the aggregates and the cytoplasm.

[0045] FIG. 5. Compound microscope fluorescence micrographs of the same
G85R SOD1 cells using GFP filter set to image the SOD1-YFP aggregates
(above) and the TRITC filter set to image the cells with the Image-iT WGA
plasma membrane dye (below).

[0049] FIG. 9. Open Field Analysis in Butaclamol-Treated G93A mice. Open
Field analysis of Wild Type littermate control mice, untreated G93A mice
and dose-response using 0.01 mg/kg, 0.1 mg/kg, and 1 mg/kg butaclamol per
day in G93A transgenic mice for distance traveled (A), resting time (B),
Ambulatory counts (C), and ambulatory time (D). There is a significant
difference between WT and untreated G93A mice, with optimal dosing at the
0.1 mg/kg dose significantly different than untreated G93A mice through
105 days and comparable (not significantly different) to WT littermate
control mice.

[0051] FIG. 11. Neuroprotective effects of butaclamol (0.1 mg/kg) in the
lumbar spinal cord in G93A transgenic ALS mice. Nissl staining of the
lumbar spinal cord from (A) wild-type mice, (B) G93A mice treated with
butaclamol, and (C) untreated G93A mice shows a marked gross atrophy in
untreated G93A mice. Treatment with butaclamol significantly improved the
gross neuropathological changes, as shown in FIGS. 3B and E. Bar in A=200
mm and is the same for B and C. High magnification photomicrographs of
Nissl staining in the ventral horn from the same sections in A, B, and C
of the lumbar spinal cord from (D) wild-type mice, (E) G93A mice treated
with butaclamol, and (F) untreated G93A mice demonstrates ventral neuron
loss and atrophy in untreated G93A mice. The treatment with butaclamol
markedly improves these neuropathological changes. Bar in D=50 pm and is
the same for E and F.

[0053] Butaclamol is a type of benzocycloheptapyridoisoquinolinol
currently in clinical use for the treatment of schizophrenia. The
structure of butaclamol contains a semi-rigid phenethylamine
pharmacophore. Butaclamol has proven to be a potent dopamine antagonist
and is often used as a reference compound in competitive screens for new
antipsychotic agents (Kukla et al., J. Med. Chem. 1979, 22(4), 401-406).
Butaclamol exists as optical isomers, with virtually all of the
dopamine-blocking activity residing in the (+) isomer. Indeed, for both
[3H] dopamine and [3H] haloperidol binding sites, (+) butaclamol is about
one hundred and one thousand times more potent, respectively, than the
clinically inactive (-) isomer (Shershow, Schizophrenia: Science and
Practice, 1978, Harvard University Press). For example, behavioral
studies done on rats with unilateral lesions in the substantia nigra have
shown that administration of 0.1 to 0.3 mg/kg of the (+) enantiomer
abolishes amphetamine-induced stereotyped and rotational behavior,
whereas the (-) enantiomer is devoid of behavioral activity even at 100
to 500 times larger than those of the (+) enantiomer. Likewise, while (+)
butaclamol antagonized epinephrine-induced mortality at high doses, (-)
butaclamol were devoid of any such activity (Voith et al., Can. J.
Physiol. Pharmacol. 1976, 54(4), 551-560). In some embodiments of the
present invention, compositions are utilized that contain and or deliver
(+) butaclamol. In some embodiments, substantially all of the butaclamol
so contained and/or delivered is (+) butaclamol. In some embodiments, at
least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,
99.8%, 99.9% or more of the butaclamol so contained and/or delivered is
(+) butaclamol.

[0054] Butaclamol may exist in several different conformations in
solution. The preferred conformations and rates of interconversion of
conformers are typically dependent on a variety of factors (e.g.,
solvent, temperature, free base or salt form of butaclamol, etc.). For
example, the HCl salt of butaclamol can exist as an equilibrium mixture
of two conformations that differ in their stereochemistry about the ring
junction containing the nitrogen atom. In DMSO, the trans form of
butaclamol HCl has a relative population of about 80%; in chloroform, the
trans form of butaclamol HCl is the only form observed. The free base of
butaclamol, in contrast to the HCl salt, interconverts much more readily,
with cis/trans conformors observable only at lower temperatures
(Casarotto et al., J. Med. Chem. 1991, 34(7), 2043-2049). Conformational
energy calculations performed on butaclamol suggest that a trans
conformer is the biologically active form that gives rise to the
neuroleptic activity of this compound under physiological conditions
(Froimowitz et al., Mol. Pharm. 1983, 24(2), 243-250).

[0055] Butaclamol compositions used to treat schizophrenia typically
contain butaclamol in the form of the free base or in the form of the HCl
salt. Common acid addition salts are typically prepared by reacting the
base form of butaclamol with the appropriate acid in an organic solvent
such as, for instance, ether or an ethanol-ether mixture. These salts,
when administered for neuroleptic purposes, often may possess comparable
pharmacologic activities as the corresponding bases. For many purposes it
is preferable to administer the salt form of butaclamol rather than the
free base. Among the acid addition salts suitable for this purpose are
salts such as the sulfate, phosphate, lactate, tartrate, maleate,
citrate, and hydrochloride (Bruderlein et al., U.S. Pat. No. 3,657,250).

[0056] As mentioned above, butaclamol has been extensively evaluated in
the treatment of chronic schizophrenic patients. In animals, this
compound exhibited a profile suggesting potential antipsychotic effects
with potential adverse effects, such as extrapyramidal and adrenergic
effects, occurring at doses substantially above the therapeutic range.
Phase I studies in healthy volunteers showed that the limiting effects in
single and repeated doses up to 32 mg/day were sedation and
extrapyramidal effects. Studies on chronic schizophrenic males and
females between the ages of 21 and 65 demonstrated that when administered
long-term at a dosage of 50 mg/day, butaclamol is indeed an active
neuroleptic agent. However, in order to minimize the extrapyramidal
effects observed at 50 mg/day, doses in the range of 5-20 mg/day were
suggested for initial treatment, and doses as low as 5-10 mg/day were
posited as reasonable maintenance doses (Clark et al., J. Clin.
Pharmacol. 1977, 17, 529).

Neurodegenerative Diseases Treated in Accordance With the Present
Invention

[0061] The present invention encompasses the recognition that butaclamol
can be effective in treating patients with amyotrophic lateral sclerosis
(ALS) or other neurodegenerative diseases characterized by the presence
of aberrant protein aggregates. Without wishing to be bound by any
particular theory or mechanism of action, methods of the invention are
useful in inhibiting or reversing abnormal protein aggregation or
reducing the toxicity of protein aggregation (e.g., SOD1 or TDP-43). The
invention provides methods for treating a subject suffering from or
susceptible to ALS or other neurodegenerative disease including the step
of administering to the subject a therapeutically effective amount of
butaclamol or a pharmaceutical composition thereof. In certain
embodiments, the subject is a transgenic mouse. In certain embodiments,
the subject is an adult human. In certain embodiments, the ALS being
treated is familial ALS. In certain embodiments, the ALS being treated is
sporadic ALS.

[0063] In some embodiments, the invention provides a method comprising
steps of administering to a subject suffering from or susceptible to ALS
an effective amount of butaclamol, such that the severity or incidence of
one or more symptoms of ALS is reduced, or its onset is delayed. In some
embodiments, butaclamol is administered in the form of a salt or
pharmaceutically acceptable composition thereof. In certain embodiments,
butaclamol is administered in accordance with the present invention to
subjects suffering from or susceptible to a neurodegenerative disease,
disorder, or condition in a form or composition and/or according to a
regimen established as useful in the treatment of schizophrenia as
discussed above. In certain embodiments, the subject suffering from or
susceptible to ALS is a human from about 40 to about 85 years of age.

[0064] In some embodiments, the ALS being treated is characterized by the
presence of abnormal protein aggregates such as, for example, SOD1
protein aggregates or TDP-43 protein aggregates. Exemplary such SOD1
protein aggregates include G93A SOD1 and G85R SOD1 protein aggregates.
Without wishing to be bound by any particular theory, use of butaclamol
in the treatment of ALS may reduce or delay the formation of such protein
aggregates.

[0065] In some embodiments, butaclamol is administered once a day. In some
embodiments, butaclamol is administered two, three, four, or five times a
day. In some embodiments, butaclamol is administered every other day. In
some embodiments, butaclamol is administered every two days. In some
embodiments, butaclamol is administered every three days. In some
embodiments, butaclamol is administered every four days. In some
embodiments, butaclamol is administered every five days. In some
embodiments, butaclamol is administered every six days. In some
embodiments, butaclamol is administered once a week. In some embodiments,
butaclamol is administered at intervals as instructed by a physician for
the duration of the life of the subject being treated. In certain
embodiments, butaclamol is administered as many times a day as necessary
to provide a therapeutically effective amount of butaclamol to treat a
subject suffering from or susceptible to ALS.

[0066] In some embodiments, the subject suffering from or susceptible to
ALS is a mammal. In some embodiments, the subject suffering from or
susceptible to ALS is a rodent, such as a rat or mouse, for example, a
mouse model of ALS. In certain embodiments, the subject suffering from or
susceptible to ALS is a human. In certain embodiments, the human is about
20, 30, 40, 50, 60, 70, 80, 90, or 100 years of age. In certain
embodiments, the human is between 40 and 85 years of age.

[0067] The efficacy of butaclamol in the treatment of neurodegenerative
diseases according to the present invention may be evaluated and followed
using any method known in the medical arts. The treatment of ALS may be
evaluated, for example, by physical examination, laboratory testing,
imaging studies, electrophysiological studies, etc. In some embodiments,
the treatment of ALS may be evaluated by monitoring the subject being
treated. In some embodiments, the subject is monitored by monitoring
motor function. In some embodiments, the subject is monitored by
monitoring body weight. In some embodiments, the subject is monitored by
monitoring survival time. In some embodiments, the subject is monitored
one, two, three, four, or five times a day. In some embodiments, the
subject is monitored one, two, three, four or five times a week. In some
embodiments, the subject is monitored twice a week. In some embodiments,
monitoring is continuous. In some embodiments, monitoring occurs for the
duration of the subject's life. In certain embodiments, the subject is
monitored one, two, or three times a day by monitoring body weight. In
some embodiments, the subject is a human and is monitored using any of
the methods known in the medical arts suitable for monitoring humans
suffering from or susceptible to a neurodegenerative disease such as ALS.
Exemplary such methods of monitoring include monitoring neurological
function, respiratory function (e.g., pulmonary function test), muscle
strength, speech, swallowing function, etc. In some embodiments,
monitoring may comprise checking for signs of toxicity; in certain
embodiments, toxicity is measured using any of the methods previously
developed to measure toxicity of butaclamol in patients being treated for
schizophrenia.

[0068] As noted above, in some embodiments, butaclamol used in accordance
with the present invention comprises (+) butaclamol. In certain
embodiments, the butaclamol is about 50% (+) butaclamol. In certain
embodiments, the butaclamol is about 60% (+) butaclamol. In certain
embodiments, the butaclamol is about 70% (+) butaclamol. In certain
embodiments, the butaclamol is about 80% (+) butaclamol. In certain
embodiments, the butaclamol is about 90% (+) butaclamol. In certain
embodiments, the butaclamol is about 95% (+) butaclamol. In certain
embodiments, the butaclamol is about 99% (+) butaclamol. In some
embodiments, substantially all of the butaclamol so contained and/or
delivered is (+) butaclamol. In some embodiments, at least 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or
more of the butaclamol so contained and/or delivered is (+) butaclamol.

[0069] Dosages of butaclamol utilized in accordance with the present
invention may vary with the form of administration and/or with the
particular subject being treated for ALS. In general, butaclamol is most
desirably administered at a concentration level that will afford
effective results without causing any harmful or deleterious side-effects
(e.g., extrapyramidal effects). In some embodiments, butaclamol is
administered in doses ranging from about 0.5 to about 500 mg/kg/day. In
some embodiments, butaclamol is administered in doses ranging from about
5 to about 100 mg/kg/day. In some embodiments, butaclamol is administered
in doses ranging from about 10 to about 100 mg/kg/day. In some
embodiments, butaclamol is administered in doses ranging from about 20 to
about 100 mg/kg/day. In some embodiments, butaclamol is administered in
doses ranging from about 30 to about 100 mg/kg/day. In some embodiments,
butaclamol is administered in doses ranging from about 40 to about 100
mg/kg/day. In some embodiments, butaclamol is administered in doses
ranging from about 50 to about 100 mg/kg/day. In some embodiments,
butaclamol is administered in doses ranging from about 60 to about 100
mg/kg/day. In some embodiments, butaclamol is administered in doses
ranging from about 70 to about 100 mg/kg/day. In some embodiments,
butaclamol is administered in doses ranging from about 80 to about 100
mg/kg/day. In some embodiments, butaclamol is administered in doses
ranging from about 90 to about 100 mg/kg/day. In some embodiments,
butaclamol is administered in doses of less than about 20 mg/day. In some
embodiments, butaclamol is administered in doses ranging from about 1
mg/kg/day to about 50 mg/kg/day. In some embodiments, butaclamol is
administered in doses ranging from about 1 mg/kg/day to about 40
mg/kg/day. In some embodiments, butaclamol is administered in doses
ranging from about 1 mg/kg to about 30 mg/kg/day. In some embodiments,
butaclamol is administered in doses ranging from about 1 mg/kg/day to
about 20 mg/kg/day. In some embodiments, butaclamol is administered in
doses ranging from about 1 mg/kg/day to about 10 mg/kg/day. In some
embodiments, butaclamol is administered in doses ranging from about 10
mg/kg/day to about 50 mg/kg/day. In some embodiments, butaclamol is
administered in doses ranging from about 10 mg/kg/day to about 40
mg/k/day. In some embodiments, butaclamol is administered in doses
ranging from about 10 mg/kg/day to about 30 mg/kg/day. In some
embodiments, butaclamol is administered in doses ranging from about 10
mg/kg/day to about 20 mg/kg/day. In some embodiments, butaclamol is
administered in doses ranging from about 20 mg/kg/day to about 50
mg/kg/day. In some embodiments, butaclamol is administered in doses
ranging from about 20 mg/kg/day to about 40 mg/kg/day. In some
embodiments, butaclamol is administered in doses ranging from about 20
mg/kg/day to about 30 mg/kg/day. In some embodiments, butaclamol is
administered in doses ranging about 25 mg/kg/day to about 50 mg/kg/day.
In some embodiments, butaclamol is administered in doses ranging about 30
mg/kg/day to about 50 mg/kg/day. In some embodiments, butaclamol is
administered in doses ranging from about 30 mg/kg/day to about 40
mg/kg/day. In some embodiments, butaclamol is administered in doses
ranging from about 40 mg/kg/day to about 50 mg/kg/day. In some
embodiments, butaclamol is administered in doses less than about 10
mg/kg/day. In some embodiments, butaclamol is administered in doses less
than about 5 mg/kg/day. In some embodiments, butaclamol is administered
in doses less than about 2 mg/kg/day. In some embodiments, butaclamol is
administered in doses less than about 1 mg/kg/day. In some embodiments,
butaclamol is administered in doses less than about 0.1 mg/kg/day. In
some embodiments, butaclamol is administered in doses less than about
0.01 mg/kg/day. In some embodiments, butaclamol is administered in doses
less than about 0.001 mg/kg/day. In some embodiments, butaclamol is
administered in doses of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,
0.9, or 1.0 mg/kg/day. In some embodiments, butaclamol is administered in
doses of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, or 0.09
mg/kg/day. In some embodiments, butaclamol is administered in doses
ranging from about 0.1 mg/kg/day to about 1 mg/kg/day. In some
embodiments, butaclamol is administered in doses ranging from about 0.01
mg/kg/day to about 0.1 mg/kg/day. In some embodiments, butaclamol is
administered in doses ranging from about 0.001 mg/kg/day to about 0.01
mg/kg/day. In some embodiments, butaclamol is administered to a subject
in doses that do not produce extrapyramidal effects in the subject. In
some embodiments, doses described above used in the treatment of
schizophrenia are in accordance with the present invention. In some
embodiments, butaclamol is administered to a subject in doses lower than
those used in the treatment schizophrenia.

[0070] In some embodiments, butaclamol is administered systemically in any
one of the doses described herein and suitable for the treatment of ALS.
Systemic administration may comprise enteral or parenteral
administration. In certain embodiments, systemic administration comprises
oral administration in solid or solution form in any one of the doses
described herein. In certain embodiments, butaclamol is administered
parenterally in any one of the doses described herein. In certain
embodiments, butaclamol is administered intraperitoneally in any one of
the doses described herein and the subject is a mouse or rat with ALS. In
some embodiments, butaclamol is administered orally and the subject is a
human with ALS.

[0071] In some embodiments, the invention provides a method comprising
steps of administering to a subject suffering from or susceptible to
abnormal protein aggregation an amount of butaclamol sufficient to reduce
or delay such abnormal protein aggregation. In certain embodiments,
reduction or inhibition of abnormal protein aggregation occurs in vivo in
a subject with ALS or another neurodegenerative disease characterized by
aberrant protein aggregation (e.g., Huntington's disease, prion disease,
Alzheimer's disease, or frontotemporal lobar degeneration). In some
embodiments, the subject is a mammal. In some embodiments, the subject is
a mouse or rat. In some embodiments, the subject is a human. In certain
embodiments, the human is about 20, 30, 40, 50, 60, 70, 80, 90, or 100
years of age. In some embodiments, abnormal protein aggregation comprises
SOD1 protein aggregates. In certain embodiments, abnormal protein
aggregation comprises G93A SOD1 protein aggregates. In certain
embodiments, abnormal protein aggregation comprises G85R SOD1 protein
aggregates. In certain embodiments, abnormal protein aggregation
comprises TDP-43 protein aggregates.

[0072] In some embodiments, butaclamol is administered to a subject with a
neurodegenerative disease using any method of administration known in the
medical arts. In certain embodiments, butaclamol may be administered
orally. In certain embodiments, butaclamol may be administered
parenterally. In certain embodiments, butaclamol is administered
intraperitoneally.

[0073] In some embodiments, the invention provides a method comprising the
steps of administering to a cell in vitro an amount of butaclamol
effective to inhibit or reverse the toxic effect of abnormal protein
aggregation. In certain embodiments, contact occurs in vitro, and the
cell is derived from a mammalian cell line. In certain embodiments,
contact occurs in vitro, and the cell is derived from a PC12 cell line.
In certain embodiments, PC12 cells may additionally contain a detectable
moiety to measure the extent of inhibition of aggregation. In certain
embodiments, a detectable moiety is associated with a protein (e.g., a
type of SOD1 protein, such as G93A SOD1 and/or G85R SOD1, or a type of
TDP-43 protein). In certain embodiments, the detectable moiety is a
fluorescent moiety (e.g., a YFP tag). In some embodiments, the detectable
moiety is a phosphorescent moiety, a radiolabel, or any other detectable
moiety known in the art, and may be detected using any of the methods
known in the art. In some embodiments, the detectable moiety may be
detected using a high content microscopy system to allow for
high-throughput screening. In certain embodiments, the detectable moiety
allows for the measurement of cell viability.

[0074] In some embodiments, the invention provides a method comprising the
steps of administering to a cell in vitro an amount of butaclamol
effective to protect against aggregated SOD1. In certain embodiments,
protection occurs in vitro in a cell culture. In some embodiments,
compounds of the invention are contacted with a cell line in vitro and
the cell line is a mammalian cell line. In certain embodiments, the cell
line is the PC12 cell line. In some embodiments, cells are associate with
a detectable moiety such as those described above. In some embodiments,
cells contain a protein labeled with a detectable moiety. In certain
embodiments, the protein is SOD1 (e.g., G93A SOD1 and/or G85R SOD1) and
the detectable moiety is a fluorescent moiety. In certain embodiments,
the detectable moiety is a fluorescent moiety (e.g., a YFP tag) that may
be detected using a high content microscopy system to allow for
high-throughput screening. In some embodiments, the detectable moiety is
a phosphorescent moiety, an epitope, radiolabel, or any other detectable
moiety known in the art, and may be detected using any of the methods
known in the art. In certain embodiments, the detectable moiety allows
for the measurement of cell viability.

[0075] In some embodiments, the invention provides a method comprising the
steps of administering to a cell in vitro an amount of butaclamol
effective to modulate proteasome function. In certain embodiments, the
cell is derived from a mammalian cell line. In some embodiments, the cell
is derived from a PC12 cell line or a HeLa cell line. In certain
embodiments, the cells contain a detectable moiety to measure the extent
to which proteasome activity is inhibited. In certain embodiments, the
protein is SOD1 (e.g., G93A SOD1 and/or G85R SOD1) and the detectable
moiety is a fluorescent moiety. In certain embodiments, the protein is
TDP-43. In certain embodiments, the detectable moiety is a fluorescent
moiety such as a Ubi-YFP tag. In some embodiments, the detectable moiety
is a Ubi-YFP tag and is detectable by fluorescence microscopy. In some
embodiments, the detectable moiety is a phosphorescent moiety, an
epitope, a radiolabel, or any other detectable moiety known in the art,
and may be detected using any of the methods known in the art. In some
embodiments, the detectable moiety may be detected using a high content
microscopy system to allow for high-throughput screening. In certain
embodiments, cell viability is measured.

[0076] In some embodiments, the present invention provides systems,
methods, and/or reagents to characterize butaclamol compounds and
compositions. In some embodiments, the present invention provides assays
to identify forms of butaclamol compounds and compositions that protect
against protein aggregate-induced cytotoxicity. In certain embodiments,
the assays are cell protection assays. Cell protection assays may used to
identify butaclamol compounds and compositions that protect cells from
the cytotoxic effects of aberrant protein aggregation. In some
embodiments, the assays are protein aggregation inhibition assays that
are used to identify butaclamol compounds and compositions that inhibit
protein aggregation in a cell or in vitro.

[0077] In some embodiments, the present invention provides a method of
identifying butaclamol compounds and compositions that protect against
protein aggregate-induced cytotoxicity comprising contacting a cell
expressing SOD1, TDP-43, or another protein susceptible to aggregation
with a test compound, incubating the cell with the test compound under
suitable conditions for an amount of time sufficient to observe a
protective effect against protein aggregate-induced cytotoxicity, and
then measuring viability in the cells treated with the test compound. In
some embodiments, the extent of protein aggregation-induced cytotoxicity
is measured by determining the level of a detectable moiety (e.g., a
fluorescent moiety) in the cell.

[0078] In certain embodiments, the expressed protein in the cell used in
the assay is a mutant SOD1 protein. In certain embodiments, the expressed
protein the cell used in the assay is a mutant TDP-43 protein. In some
embodiments, the expressed protein is SOD1 protein associated with a
detectable moiety. In certain embodiments, the expressed protein is a
fluorescently tagged mutant SOD1 protein, and the fluorescent moiety is a
YFP tag. In some embodiments, the detectable moiety is a phosphorescent
moiety, epitope, or radiolabel. In some embodiments, the detectable
moiety is any suitable detectable moiety known to those or ordinary skill
in the art and may be detected using any method known in the art. In some
embodiments, the detectable moiety is a fluorescent tag (e.g., a YFP tag)
that can be detected with a high content microscopy system. In some
embodiments, the high content microscopy system detects cell viability
and facilitates high-throughput screening of a plurality of butaclamol
compounds.

[0079] Cells may be pre-treated with an agent that modulates the
expression of a protein of interest (e.g., SOD1, TDP-43) in the assay.
The agent may, for instance, induce the expression of a gene responsible
for the protein of interest (e.g., doxycycline-inducible promoter). In
some embodiments, cells may also be treated with an agent that modulates
proteasome activity. In certain embodiments, the agent may be a
proteasome inhibitor (e.g., MG132). In some embodiments, cell viability
of cells pre-treated with an agent described herein is measured using
methods described above.

[0080] In certain embodiments, the time of incubation of a cell with a
butaclamol compound or composition ranges from approximately 1 minute to
approximately 1 week. In some embodiments, the time of incubation ranges
from approximately 5 minutes to approximately 1 week. In some
embodiments, the time of incubation ranges from approximately 30 minutes
to approximately 2 days. In some embodiments, the time of incubation
ranges from approximately 30 minutes to approximately 1 day. In some
embodiments, the time of incubation ranges from approximately 1 hour to
approximately 1 day. In some embodiments, the time of incubation ranges
from approximately 1 hour to approximately 18 hours. In some embodiments,
the time of incubation ranges from approximately 1 hour to approximately
12 hours. In some embodiments, the time of incubation ranges from
approximately 1 hour to approximately 6 hours. In some embodiments, the
time of incubation ranges from approximately 1 hour to approximately 3
hours. In some embodiments, the time of incubation is approximately 6
hours. In some embodiments, the time of incubation is approximately 12
hours. In some embodiments, the time of incubation is approximately 18
hours. In some embodiments, the time of incubation is approximately 24
hours.

[0081] In certain embodiments, the temperature during incubation of a cell
with a butaclamol compound or composition ranges from approximately
20° C. to approximately 45° C. In certain embodiments, the
temperature ranges from approximately 20° C. to approximately
40° C. In certain embodiments, the temperature ranges from
approximately 25° C. to approximately 40° C. In certain
embodiments, the temperature ranges from approximately 30° C. to
approximately 40° C. In certain embodiments, the temperature is
approximately 30° C. In certain embodiments, the temperature is
approximately 37° C.

[0082] Butaclamol compounds or compositions that are active in the
above-mentioned assay could theoretically protect against abnormal
protein aggregate-induced cytotoxicity through a number of biological
mechanisms. The present invention additionally provides methods to screen
for butaclamol compounds or compositions that protect against abnormal
protein-aggregate induced cytotoxicity wherein the protein aggregation is
inhibited in a non-specific manner.

[0083] Butaclamol compounds or compositions which inhibit aberrant protein
aggregation can be identified using methods similar to those described
above in the aforementioned cytotoxicity assay. In some embodiments, the
present invention provides a method of identifying butaclamol compounds
or compositions that inhibit aberrant protein aggregation comprising
contacting a cell expressing SOD1 or other protein susceptible to
aggregation with a butaclamol compound or composition, incubating the
cell with the compound or composition under suitable conditions, and then
measuring the extent of protein aggregation in the cells treated with the
butaclamol compound or composition as compared to a control. In certain
embodiments, the extent of inhibition of protein aggregation is measured
by staining the protein aggregates with a detectable stain (e.g.,
Image-iT plasma membrane dye). In some embodiments, the detectable stain
is detected using a scanning device (e.g., Cellomics Arrayscan). In
certain embodiments, the protein aggregates are detected using any method
of detecting protein aggregates known in the art.

[0084] Butaclamol compounds and compositions identified using the
above-mentioned assays may be further examined using biological assays to
guide structure-activity relationship (SAR) analyses of the identified
compounds. Biological assays and SAR analyses are known to those of skill
in the art.

Pharmaceutical Compositions

[0085] In some embodiments, the present invention provides pharmaceutical
compositions, which comprise a therapeutically effective amount of
butaclamol, formulated together with one or more pharmaceutically
acceptable carriers (additives) and/or diluents. As described in detail,
the pharmaceutical compositions of the present invention may be specially
formulated for administration in solid or liquid form, including those
adapted for the following: oral administration, for example, drenches
(aqueous or non-aqueous solutions or suspensions), tablets, e.g., those
targeted for buccal, sublingual, and systemic absorption, boluses,
powders, granules, pastes for application to the tongue; parenteral
administration, for example, by subcutaneous, intramuscular, intravenous
or epidural injection as, for example, a sterile solution or suspension,
or sustained-release formulation; topical application, for example, as a
cream, ointment, or a controlled-release patch or spray applied to the
skin, lungs, or oral cavity; intravaginally or intrarectally, for
example, as a pessary, cream or foam; sublingually; ocularly;
transdermally; or nasally, pulmonary and to other mucosal surfaces.

[0086] The phrase "pharmaceutically acceptable" is employed herein to
refer to those compounds, materials, compositions, and/or dosage forms
which are, within the scope of sound medical judgment, suitable for use
in contact with the tissues of human beings and animals without excessive
toxicity, irritation, allergic response, or other problem or
complication, commensurate with a reasonable benefit/risk ratio.

[0087] In another aspect, the present invention provides "pharmaceutically
acceptable" compositions, which comprise a therapeutically effective
amount of one or more of the compounds described herein, formulated
together with one or more pharmaceutically acceptable carriers
(additives) and/or diluents. As described in detail, the pharmaceutical
compositions of the present invention may be specially formulated for
administration in solid or liquid form, including those adapted for the
following: oral administration, for example, drenches (aqueous or
non-aqueous solutions or suspensions), tablets, e.g., those targeted for
buccal, sublingual, and systemic absorption, boluses, powders, granules,
pastes for application to the tongue; parenteral administration, for
example, by subcutaneous, intramuscular, intravenous or epidural
injection as, for example, a sterile solution or suspension, or
sustained-release formulation; topical application, for example, as a
cream, ointment, or a controlled-release patch or spray applied to the
skin, lungs, or oral cavity; intravaginally or intrarectally, for
example, as a pessary, cream or foam; sublingually; ocularly;
transdermally; or nasally, pulmonary and to other mucosal surfaces.

[0088] The phrase "pharmaceutically acceptable carrier" as used herein
means a pharmaceutically acceptable material, composition or vehicle,
such as a liquid or solid filler, diluent, excipient, or solvent
encapsulating material, involved in carrying or transporting the subject
compound from one organ, or portion of the body, to another organ, or
portion of the body. Each carrier should be "acceptable" in the sense of
being compatible with the other ingredients of the formulation and not
injurious to the patient. Some examples of materials which can serve as
pharmaceutically acceptable carriers include: sugars, such as lactose,
glucose and sucrose; starches, such as corn starch and potato starch;
cellulose, and its derivatives, such as sodium carboxymethyl cellulose,
ethyl cellulose and cellulose acetate; powdered tragacanth; malt;
gelatin; talc; excipients, such as cocoa butter and suppository waxes;
oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,
olive oil, corn oil and soybean oil; glycols, such as propylene glycol;
polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;
esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,
such as magnesium hydroxide and aluminum hydroxide; alginic acid;
pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH
buffered solutions; polyesters, polycarbonates and/or polyanhydrides; and
other non-toxic compatible substances employed in pharmaceutical
formulations.

[0089] In some embodiments, butaclamol for use in accordance with the
present invention is provided in a salt form. These salts can be prepared
in situ in the administration vehicle or the dosage form manufacturing
process, or by separately reacting a purified compound of the invention
in its free base form with a suitable organic or inorganic acid, and
isolating the salt thus formed during subsequent purification.
Representative salts include salts such as the sulfate, phosphate,
lactate, tartrate, maleate, citrate, hydrochloride (Bruderlein et al.,
U.S. Pat. No. 3,657,250) and the like. See also, for example, Berge et
al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19; incorporated
herein by reference.

[0090] Wetting agents, emulsifiers and lubricants, such as sodium lauryl
sulfate and magnesium stearate, as well as coloring agents, release
agents, coating agents, sweetening, flavoring and perfuming agents,
preservatives and antioxidants can also be present in the compositions.

[0092] Formulations of the present invention include those suitable for
oral, nasal, topical (including buccal and sublingual), rectal, vaginal
and/or parenteral administration. The formulations may conveniently be
presented in unit dosage form and may be prepared by any methods well
known in the art of pharmacy. The amount of active ingredient which can
be combined with a carrier material to produce a single dosage form will
vary depending upon the host being treated, and the particular mode of
administration. The amount of active ingredient that can be combined with
a carrier material to produce a single dosage form will generally be that
amount of butaclamol which produces a therapeutic effect. Generally, this
amount will range from about 1% to about 99% of active ingredient,
preferably from about 5% to about 70%, most preferably from about 10% to
about 30%.

[0093] In certain embodiments, a formulation of the present invention
comprises an excipient selected from the group consisting of
cyclodextrins, liposomes, micelle forming agents, e.g., bile acids, and
polymeric carriers, e.g., polyesters and polyanhydrides; and a compound
of the present invention. In certain embodiments, an aforementioned
formulation renders orally bioavailable a compound of the present
invention.

[0094] Methods of preparing these formulations or compositions include the
step of bringing into association a compound of the present invention
with the carrier and, optionally, one or more accessory ingredients. In
general, the formulations are prepared by uniformly and intimately
bringing into association a compound of the present invention with liquid
carriers, or finely divided solid carriers, or both, and then, if
necessary, shaping the product.

[0095] Formulations of the invention suitable for oral administration may
be in the form of capsules, cachets, pills, tablets, lozenges (using a
flavored basis, usually sucrose and acacia or tragacanth), powders,
granules, or as a solution or a suspension in an aqueous or non-aqueous
liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an
elixir or syrup, or as pastilles (using an inert base, such as gelatin
and glycerin, or sucrose and acacia) and/or as mouth washes and the like,
each containing a predetermined amount of a compound of the present
invention as an active ingredient. A compound of the present invention
may also be administered as a bolus, electuary or paste.

[0096] In solid dosage forms of the invention for oral administration
(capsules, tablets, pills, dragees, powders, granules and the like), the
active ingredient is mixed with one or more pharmaceutically-acceptable
carriers, such as sodium citrate or dicalcium phosphate, and/or any of
the following: fillers or extenders, such as starches, lactose, sucrose,
glucose, mannitol, and/or silicic acid; binders, such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
sucrose and/or acacia; humectants, such as glycerol; disintegrating
agents, such as agar-agar, calcium carbonate, potato or tapioca starch,
alginic acid, certain silicates, and sodium carbonate; solution retarding
agents, such as paraffin; absorption accelerators, such as quaternary
ammonium compounds; wetting agents, such as, for example, cetyl alcohol,
glycerol monostearate, and non-ionic surfactants; absorbents, such as
kaolin and bentonite clay; lubricants, such as talc, calcium stearate,
magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,
and mixtures thereof; and coloring agents. In the case of capsules,
tablets and pills, the pharmaceutical compositions may also comprise
buffering agents. Solid compositions of a similar type may also be
employed as fillers in soft and hard-shelled gelatin capsules using such
excipients as lactose or milk sugars, as well as high molecular weight
polyethylene glycols and the like.

[0097] A tablet may be made by compression or molding, optionally with one
or more accessory ingredients. Compressed tablets may be prepared using
binder (for example, gelatin or hydroxypropylmethyl cellulose),
lubricant, inert diluent, preservative, disintegrant (for example, sodium
starch glycolate or cross-linked sodium carboxymethyl cellulose),
surface-active or dispersing agent. Molded tablets may be made in a
suitable machine in which a mixture of the powdered compound is moistened
with an inert liquid diluent.

[0098] The tablets, and other solid dosage forms of the pharmaceutical
compositions of the present invention, such as dragees, capsules, pills
and granules, may optionally be scored or prepared with coatings and
shells, such as enteric coatings and other coatings well known in the
pharmaceutical-formulating art. They may also be formulated so as to
provide slow or controlled release of the active ingredient therein
using, for example, hydroxypropylmethyl cellulose in varying proportions
to provide the desired release profile, other polymer matrices, liposomes
and/or microspheres. They may be formulated for rapid release, e.g.,
freeze-dried. They may be sterilized by, for example, filtration through
a bacteria-retaining filter, or by incorporating sterilizing agents in
the form of sterile solid compositions that can be dissolved in sterile
water, or some other sterile injectable medium immediately before use.
These compositions may also optionally contain opacifying agents and may
be of a composition that they release the active ingredient(s) only, or
preferentially, in a certain portion of the gastrointestinal tract,
optionally, in a delayed manner. Examples of embedding compositions that
can be used include polymeric substances and waxes. The active ingredient
can also be in micro-encapsulated form, if appropriate, with one or more
of the above-described excipients.

[0102] Formulations of the pharmaceutical compositions of the invention
for rectal or vaginal administration may be presented as a suppository,
which may be prepared by mixing one or more compounds of the invention
with one or more suitable nonirritating excipients or carriers
comprising, for example, cocoa butter, polyethylene glycol, a suppository
wax or a salicylate, and which is solid at room temperature, but liquid
at body temperature and, therefore, will melt in the rectum or vaginal
cavity and release the active compound.

[0103] Dosage forms for the topical or transdermal administration of
butaclamol include powders, sprays, ointments, pastes, creams, lotions,
gels, solutions, patches and inhalants. The butaclamol may be mixed under
sterile conditions with a pharmaceutically-acceptable carrier, and with
any preservatives, buffers, or propellants which may be required.

[0105] Powders and sprays can contain, in addition to butaclamol,
excipients such as lactose, talc, silicic acid, aluminum hydroxide,
calcium silicates and polyamide powder, or mixtures of these substances.
Sprays can additionally contain customary propellants, such as
chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as
butane and propane.

[0106] Transdermal patches may have the added advantage of providing
controlled delivery of butaclamol to the body. Dissolving or dispersing
butaclamol in the proper medium can make such dosage forms. Absorption
enhancers can also be used to increase the flux of butaclamol across the
skin. Either providing a rate controlling membrane or dispersing
butaclamol in a polymer matrix or gel can control the rate of such flux.

[0107] Ophthalmic formulations, eye ointments, powders, solutions and the
like, are also contemplated as being within the scope of this invention.

[0108] Pharmaceutical compositions of this invention suitable for
parenteral administration comprise butaclamol in combination with one or
more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous
solutions, dispersions, suspensions or emulsions, or sterile powders
which may be reconstituted into sterile injectable solutions or
dispersions just prior to use, which may contain sugars, alcohols,
antioxidants, buffers, bacteriostats, solutes which render the
formulation isotonic with the blood of the intended recipient or
suspending or thickening agents.

[0109] Examples of suitable aqueous and nonaqueous carriers, which may be
employed in the pharmaceutical compositions of the invention include
water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene
glycol, and the like), and suitable mixtures thereof, vegetable oils,
such as olive oil, and injectable organic esters, such as ethyl oleate.
Proper fluidity can be maintained, for example, by the use of coating
materials, such as lecithin, by the maintenance of the required particle
size in the case of dispersions, and by the use of surfactants.

[0110] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing agents.
Prevention of the action of microorganisms upon the butaclamol may be
ensured by the inclusion of various antibacterial and antifungal agents,
for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It
may also be desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition, prolonged
absorption of the injectable pharmaceutical form may be brought about by
the inclusion of agents which delay absorption such as aluminum
monostearate and gelatin.

[0111] In some cases, in order to prolong the effect of a drug, it is
desirable to slow the absorption of the drug from subcutaneous or
intramuscular injection. This may be accomplished by the use of a liquid
suspension of crystalline or amorphous material having poor water
solubility. The rate of absorption of the drug then depends upon its rate
of dissolution, which in turn, may depend upon crystal size and
crystalline form. Alternatively, delayed absorption of a
parenterally-administered drug form is accomplished by dissolving or
suspending the drug in an oil vehicle.

[0112] Injectable depot forms are made by forming microencapsule matrices
of the subject compounds in biodegradable polymers such as
polylactide-polyglycolide. Depending on the ratio of drug to polymer, and
the nature of the particular polymer employed, the rate of drug release
can be controlled. Examples of other biodegradable polymers include
poly(orthoesters) and poly(anhydrides). Depot injectable formulations are
also prepared by entrapping the drug in liposomes or microemulsions,
which are compatible with body tissue.

[0113] In certain embodiments, butaclamol or pharmaceutical preparation is
administered orally. In other embodiments, butaclamol or pharmaceutical
preparation is administered intravenously. Alternative routs of
administration include sublingual, intramuscular, and transdermal
administrations.

[0114] When butaclamol is administered as a pharmaceutical, to humans and
animals, it can be given per se or as a pharmaceutical composition
containing, for example, 0.1% to 99.5% (more preferably, 0.5% to 90%) of
active ingredient in combination with a pharmaceutically acceptable
carrier.

[0115] The preparations of the present invention may be given orally,
parenterally, topically, or rectally. Butaclamol is of course given in
forms suitable for each administration route. For example, it may be
administered in tablets or capsule form, by injection, inhalation, eye
lotion, ointment, suppository, etc. administration by injection, infusion
or inhalation; topical by lotion or ointment; and rectal by
suppositories. Oral administrations are preferred.

[0117] The phrases "systemic administration," "administered systemically,"
"peripheral administration," and "administered peripherally" as used
herein mean the administration of a compound, drug or other material
other than directly into the central nervous system, such that it enters
the patient's system and, thus, is subject to metabolism and other like
processes, for example, subcutaneous administration.

[0118] Butaclamol may be administered to humans and other animals for
therapy by any suitable route of administration, including orally,
nasally, as by, for example, a spray, rectally, intravaginally,
parenterally, intracisternally and topically, as by powders, ointments or
drops, including buccally and sublingually.

[0119] Regardless of the route of administration selected, butaclamol,
which may be used in a suitable hydrated form, and/or the pharmaceutical
compositions of the present invention, is formulated into
pharmaceutically-acceptable dosage forms by conventional methods known to
those of skill in the art.

[0120] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of butaclamol may be varied so as to obtain
an amount of the active ingredient that is effective to achieve the
desired therapeutic response for a particular patient, composition, and
mode of administration, without being toxic to the patient.

[0121] The selected dosage level will depend upon a variety of factors
including the particular salt or isomer composition of butaclamol used,
the route of administration, the time of administration, the rate of
excretion or metabolism of the particular composition being employed, the
duration of the treatment, other drugs, compounds and/or materials used
in combination with butaclamol, the age, sex, weight, condition, general
health and prior medical history of the patient being treated, and like
factors well known in the medical arts.

[0122] A physician or veterinarian having ordinary skill in the art can
readily determine and prescribe the effective amount of the
pharmaceutical composition required. For example, the physician or
veterinarian could start doses of butaclamol employed in the
pharmaceutical composition at levels lower than that required to achieve
the desired therapeutic effect and then gradually increasing the dosage
until the desired effect is achieved.

[0123] In some embodiments, butaclamol or a pharmaceutical composition
thereof is provided to a synucleinopathic subject (e.g., a subject with
ALS) chronically. Chronic treatments include any form of repeated
administration for an extended period of time, such as repeated
administrations for one or more months, between a month and a year, one
or more years, or longer. In many embodiments, a chronic treatment
involves administering butaclamol or a pharmaceutical composition thereof
repeatedly over the life of the subject. Preferred chronic treatments
involve regular administrations, for example one or more times a day, one
or more times a week, or one or more times a month. In general, a
suitable dose such as a daily dose of butaclamol will be that amount of
butaclamol that is the lowest dose effective to produce a therapeutic
effect. Such an effective dose will generally depend upon the factors
described above. Generally doses of butaclamol for a patient, when used
for the indicated effects, will range from about 0.0001 to about 100 mg
per kg of body weight per day. In some embodiments, the daily dosage will
range from about 1 to about 50 mg of compound per kg of body weight. In
certain embodiments, the daily dosage will range from about 25 to about
50 mg of compound per kg of body weight. However, lower or higher doses
can be used. In some embodiments, the dose administered to a subject may
be modified as the physiology of the subject changes due to age, disease
progression, weight, or other factors.

[0124] If desired, the effective daily dose of the active compound may be
administered as two, three, four, five, six, or more sub-doses
administered separately at appropriate intervals throughout the day,
optionally, in unit dosage forms.

[0125] While it is possible for butaclamol to be administered alone, it is
preferable to administer it as a pharmaceutical formulation (composition)
as described above.

[0126] Butaclamol may be formulated for administration in any convenient
way for use in human or veterinary medicine, by analogy with other
pharmaceuticals.

[0127] According to the invention, butaclamol can be formulated or
administered using methods that help the it cross the blood-brain barrier
(BBB) to a greater extent than would occur without a particular
formulation of route of administration. The vertebrate brain (and CNS)
has a unique capillary system unlike that in any other organ in the body.
The unique capillary system has morphologic characteristics which make up
the blood-brain barrier (BBB). The blood-brain barrier acts as a
system-wide cellular membrane that separates the brain interstitial space
from the blood. The unique morphologic characteristics of the brain
capillaries that make up the BBB are: (a) epithelial-like high resistance
tight junctions which literally cement all endothelia of brain
capillaries together, and (b) scanty pinocytosis or transendothelial
channels, which are abundant in endothelia of peripheral organs.

[0128] Antibodies are another method for delivery of compositions of the
invention. For example, an antibody that is reactive with a transferrin
receptor present on a brain capillary endothelial cell, can be conjugated
to a neuropharmaceutical agent to produce an antibody-neuropharmaceutical
agent conjugate (U.S. Pat. No. 5,004,697, incorporated herein in its
entirety by reference). The method is conducted under conditions whereby
the antibody binds to the transferrin receptor on the brain capillary
endothelial cell and the neuropharmaceutical agent is transferred across
the blood brain barrier in a pharmaceutically active form. The uptake or
transport of antibodies into the brain can also be greatly increased by
cationizing the antibodies to form cationized antibodies having an
isoelectric point of between about 8.0 to 11.0 (U.S. Pat. No. 5,527,527,
incorporated herein in its entirety by reference).

[0129] A ligand-neuropharmaceutical agent fusion protein is another method
useful for delivery of compositions to a host (U.S. Pat. No. 5,977,307,
incorporated herein in its entirety by reference). The ligand is reactive
with a brain capillary endothelial cell receptor. The method is conducted
under conditions whereby the ligand binds to the receptor on a brain
capillary endothelial cell and the neuropharmaceutical agent is
transferred across the blood brain barrier in a pharmaceutically active
form. In some embodiments, a ligand-neuropharmaceutical agent fusion
protein, which has both ligand binding and neuropharmaceutical
characteristics, can be produced as a contiguous protein by using genetic
engineering techniques. Gene constructs can be prepared comprising DNA
encoding the ligand fused to DNA encoding the protein, polypeptide or
peptide to be delivered across the blood brain barrier. The ligand coding
sequence and the agent coding sequence are inserted in the expression
vectors in a suitable manner for proper expression of the desired fusion
protein. The gene fusion is expressed as a contiguous protein molecule
containing both a ligand portion and a neuropharmaceutical agent
portion).

[0130] The permeability of the blood brain barrier can be increased by
administering a blood brain barrier agonist, for example bradykinin (U.S.
Pat. No. 5,112,596, incorporated herein in its entirety by reference), or
polypeptides called receptor mediated permeabilizers (RMP) (U.S. Pat. No.
5,268,164, incorporated herein in its entirety by reference). Exogenous
molecules can be administered to the host's bloodstream parenterally by
subcutaneous, intravenous or intramuscular injection or by absorption
through a bodily tissue, such as the digestive tract, the respiratory
system or the skin. The form in which the molecule is administered (e.g.,
capsule, tablet, solution, emulsion) depends, at least in part, on the
route by which it is administered. The administration of the exogenous
molecule to the host's bloodstream and the intravenous injection of the
agonist of blood-brain barrier permeability can occur simultaneously or
sequentially in time. For example, a therapeutic drug can be administered
orally in tablet form while the intravenous administration of an agonist
of blood-brain barrier permeability is given later (e.g., between 30
minutes later and several hours later). This allows time for the drug to
be absorbed in the gastrointestinal tract and taken up by the bloodstream
before the agonist is given to increase the permeability of the
blood-brain barrier to the drug. On the other hand, an agonist of
blood-brain barrier permeability (e.g., bradykinin) can be administered
before or at the same time as an intravenous injection of a drug. Thus,
the term "co-administration" is used herein to mean that the agonist of
blood-brain barrier and the exogenous molecule will be administered at
times that will achieve significant concentrations in the blood for
producing the simultaneous effects of increasing the permeability of the
blood-brain barrier and allowing the maximum passage of the exogenous
molecule from the blood to the cells of the central nervous system).

[0131] In other embodiments, butaclamol can be formulated as a prodrug
with a fatty acid carrier (and optionally with another neuroactive drug).
The prodrug is stable in the environment of both the stomach and the
bloodstream and may be delivered by ingestion. The prodrug passes readily
through the blood brain barrier. The prodrug preferably has a brain
penetration index of at least two times the brain penetration index of
the drug alone. Once in the central nervous system, the prodrug, which
preferably is inactive, is hydrolyzed into the fatty acid carrier and the
butaclamol (and optionally another drug). The carrier preferably is a
normal component of the central nervous system and is inactive and
harmless. Butaclamol, once released from the fatty acid carrier, is
active. Preferably, the fatty acid carrier is a partially-saturated
straight chain molecule having between about 16 and 26 carbon atoms, and
more preferably 20 and 24 carbon atoms. Examples of fatty acid carriers
are provided in U.S. U.S. Pat. Nos. 4,939,174; 4,933,324; 5,994,932;
6,107,499; 6,258,836; and 6,407,137, the disclosures of which are
incorporated herein by reference in their entirety.).

[0132] The administration butaclamol or pharmaceutical compositions
thereof may be for either prophylactic or therapeutic purposes. When
provided prophylactically, butaclamol is provided in advance of disease
symptoms. The prophylactic administration of butaclamol serves to prevent
or reduce the rate of onset of symptoms of ALS. When provided
therapeutically, the butaclamol is provided at (or shortly after) the
onset of the appearance of symptoms of actual disease. In some
embodiments, the therapeutic administration of butaclamol serves to
reduce the severity and duration of the disease.

[0133] The function and advantage of these and other embodiments of the
present invention will be more fully understood from the examples
described below. The following examples are intended to illustrate the
benefits of the present invention, but do not exemplify the full scope of
the invention.

EXAMPLES

[0134] The foregoing has been a description of certain non-limiting
embodiments of the invention. Accordingly, it is to be understood that
the embodiments of the invention herein described are merely illustrative
of the application of the principles of the invention. Reference herein
to details of the illustrated embodiments is not intended to limit the
scope of the claims.

Example 1

Assays for Identification of Compounds that Protect Against Mutant
SOD1--Induced Cytotoxicity

High Throughput Assays

[0135] Cultured cells are utilized to conduct high throughput assays for
compounds that protect against mutant SOD1-induced cytotoxicity. Two
assays are used: first, in the cytotoxicity protection assay, compounds
are screened for their ability to protect cells from the cytotoxic
effects of aggregated mutant SOD1, irrespective of mechanism of drug
action. Second, in the protein aggregation assay, compounds are screened
for their ability to reduce aggregation of mutant SOD1. The high
throughput cytotoxicity protection assay is the primary screen and
compounds active in the primary screen (and their analogs) move forward
into the secondary screen for protein aggregation.

[0136] The high throughput cytotoxicity protection assay was carried out
in PC12 cells that express mutant G93A SOD1 as a YFP fusion protein from
a doxycycline-inducible promoter (Matsumoto et al., J. Cell. Biol. 2005,
171, 75). Several lines of evidence suggest that cytotoxicity of protein
aggregates in ALS is due at least in part to inhibition of the proteasome
(Bruijin et al. Annu. Rev. Neurosci. 2004, 27, 723-729; Cleveland et al.
Nat. Rev. Neurosci. 2001, 2(11), 806). This idea was tested by examining
the sensitivity of PC12 cells to SOD1 aggregates in the presence and
absence of proteasome inhibitor MG132. PC12 cells expressing no SOD1,
wild type SOD1, G85R SOD1 or G93A SOD1 were grown with or without MG132
(FIG. 1). Cells expressing no SOD1, wild type SOD1 and G85R SOD1 were
relatively insensitive to MG132, with an IC50 of approximately 400
nM. In contrast, cells expressing G93A SOD1 were approximately 5-fold
more sensitive to MG132 (IC50˜75 nM). In these cells, protein
aggregation was detected after 24 h and loss of cell viability was
detected at approximately 48 h. Qualitatively similar results were
obtained with the structurally distinct proteasome inhibitor bortezomib
(Velcade®), suggesting that PC12 cells are indeed susceptible to
proteasome inhibition and not some other effect of MG132. The ability of
protein aggregates to induce cell death was examined by treating G93A
SOD1-expressing cells with MG132 for 24 h, removing the MG132 by washing
and assaying cell viability after another 24 h. Because the loss of cell
viability was similar following MG132 removal (FIG. 1, part C), it is
likely that mutant SOD1 aggregates contribute directly to cytotoxicity in
PC12 cells. However, this effect is specific for G93A SOD1 suggesting
that this mutant may produce higher levels of a toxic aggregated form of
SOD1.

[0137] Based on these results, a high throughput screen was developed for
compounds that protect against the cytotoxicity of G93A SOD1 protein
aggregates using geldanamycin or radicicol as a positive control. PC12
cells expressing G93A SOD1 were treated with 100 nM MG132 with or without
co-treatment with geldanamycin or radicicol. The latter compounds inhibit
the chaperone HSP90 and induce expression of other chaperones. As
anticipated, radicicol reduced formation of protein aggregates and
increased cell viability in a dose-dependent manner (FIG. 2). Statistical
analysis of the data produced a Z' value of 0.55, which would predict
good performance as a positive control in a high throughput screen (Zhang
et al. J. Biomol. Screen 1999, 4(2), 67-73).

Mutant SOD1 Direct Protein Aggregation Assay.

[0138] Compounds that are active in the above assay could theoretically
protect against mutant SOD1-induced cytotoxicity through a number of
mechanisms, including the following: 1) Compounds could nonspecifically
block or reverse protein aggregation via chaperone induction, as observed
for radicicol and geldanamycin 2) Compounds could block or reverse the
aggregation of a specific aggregated protein form 3) Compounds could
interfere with an event downstream of protein aggregation that plays a
critical role in mutant SOD1-induced cytotoxicity (e.g., proteasome
function). 4) Compounds could act directly on SOD1 in a manner that
prevents mutant SOD1 aggregation. These possibilities were tested using
an assay that directly measures protein aggregation. In addition, unlike
the high throughput cytotoxicity protection assay, the protein
aggregation assay is based on G85R SOD1; this broadens the scope of the
screening strategy, and should eliminate compounds with highly specific
activity (i.e., G93A SOD1 limited) against protein aggregation.

[0139] In PC12 cells that express wild-type SOD1, SOD1 was diffusely
localized throughout the cell (Matsumoto et al. J. Cell. Biol 2005,
171(1), 75-85). In contrast, G85R SOD1 showed heterogeneous patterns of
localization; in most cells, G85R was diffusely localized throughout the
cell, but in ˜5% of the cells, G85R SOD1 was localized in large
peri-nuclear aggregates. In cells treated with MG132, up to 75% of cells
expressing G85R SOD1 contain such protein aggregates (FIG. 3), but no
aggregation was observed in cells expressing wild-type SOD1. Cells
expressing G93A mutant SOD1 showed an intermediate level of protein
aggregation: none of the cells developed protein aggregates in the
absence of MG132, and ˜75% of the cells had protein aggregates
following treatment with MG132 (FIG. 3). Similar effects were observed in
cells treated with bortezomib (Velcade®). Therefore, these effects
are likely to be due to MG132-induced proteasome inhibition, and not due
to an off-target effect of MG132.

[0140] The sensitivity of this assay was optimized by selecting conditions
that maximize the difference between active and inactive samples. The
identification of a positive control is a crucial step in assay
development. Thus, PC12 cells expressing G85R or G93A mutant SOD1 were
treated with MG132 to induce protein aggregation, and then co-treated
with candidate chemical suppressors of protein aggregation. Two compounds
with similar activity were identified in these experiments: geldanamycin
and radicicol. Both compounds induce heat shock transcription factor
HSF-1, which in turn induces the heat shock response (FIG. 4). Treatment
with radicicol reduced the proportion of cells with aggregates from 75%
to 25%, a sufficient difference to allow visual scoring for compounds
with efficacy equal to or greater than radicicol.

[0141] To allow this assay to be used in a high-throughput manner, a
Cellomics Arrayscan® high content microscopy system was used for
screening and quantification. Initial experiments indicated that G85R
SOD1 aggregates were more readily recognized by the high content
microscopy system and its computer algorithm. Because the most robust
high content assays measure events on a per cell basis, it was necessary
to select a fluorescent stain that marks whole cells to be used with a
compatible stain that marks intracellular structures. On the basis of
pilot experiments with a number of vital dyes, an Image-iT conjugated
wheat germ agglutinin (WGA) dye from Molecular Probes was selected and a
computer algorithm for detecting WGA was developed. As shown in FIG. 5,
WGA provided an excellent cellular marker that did not interfere with
detection of YFP-tagged SOD1.

Example 2

Identification of Butaclamol

[0142] Screening campaign results: The Cambria chemical library is
composed of >50,000 small molecule compounds including most FDA
approved drugs, a diverse collection of biochemical reagent compounds and
structurally diverse random chemistry including compounds unique to
Cambria which were sourced via special contractual arrangements. The
primary screen yielded 68 primary active compounds, including 67
structurally diverse compounds and a single clinical agent (butaclamol).
This collection of actives was then counter-screened to remove
artifactual fluorescent compounds and protein synthesis inhibitors that
could artifactually block the production of the toxic G93A SOD1 insult.
The protection actives were tested for effects on SOD1 protein
aggregation and all of these compounds except butaclamol and one
structurally novel compound were active in that assay. Chemical structure
comparisons of the active compounds grouped them into 17 chemotype
classes plus 15 singleton hits.

[0143] Neuroleptic therapeutics: Ten neuroleptic therapeutics,
representing both the `typical` and `atypical` compound types, were
screened in the protection assay (Table 1). Only butaclamol showed
activity. Butaclamol was inactive when tested in the aggregation assay.
This result suggests that butaclamol is either acting by blocking the
toxic effects of the aggregated SOD1 or is blocking the formation of a
toxic SOD1 species that is not scored/detected in the aggregation assay.

[0145] Butaclamol behaves similarly to radicicol in the protection assay,
providing incomplete protection. This is likely due to the fact that both
of these compounds are toxic at higher doses and that the observed
efficacy (maximum viability) is a combination of protective and toxic
effects. Butaclamol provides protection over a wide range of
concentrations and the plateau of efficacy declines as toxic effects
become prominent at higher drug concentrations (FIG. 6). Butaclamol is a
diasteriomer and the two different isomeric forms can be physically
separated. We therefore tested each separate enantiomer based on the
following reasoning: certain NSAID drugs including flurbiprofen will
inhibit γ-secretase and lower A1342 levels and thus have potential
therapeutic value for the treatment of Alzheimer's disease (Ericson et
al., J. Clinical Invest. (2003), 112, 440). Since flurbiprofen is a
diasteriomer, one development strategy has been to utilize the
flurbiprofen enantiomer lacking cyclooxygenase inhibitory activity as a
means to develop treatment that lack toxic gastric side effects. We took
an analogous approach with butaclamol and tested the separated
enantiomers for protective effects (FIG. 6), in case the distomer lacking
dopamine receptor binding and thus extrapyramidal effects (-butaclamol)
would be active. In this experiment we found only the eutomer
(+butaclamol) showed activity.

Example 3

Butaclamol Activity In Vivo

[0146] The present example demonstrates that butaclamol has protective
effects in the G93A SOD1 ALS mouse model at doses that do not produce
extrapyramidal effects. Butaclamol produces extrapyramidal effects in
patients at doses as low as 10 mg/day. Therefore dosing in clinical
studies should desirably be below this value. Butaclamol has a molecular
weight of 362 and assuming a volume of distribution equal to total body
water (˜60% of weight), a 10 mg dose could yield a potential
maximum concentration of ˜0.65 μM in a 70 kg man. This dose is
approximately twice the ED50 for butaclamol in the protection assay,
which might suggest that only minimal protective effects is achieved
without limiting side effects (i.e., that there might be a narrow
therapeutic window). However, the protection assay is a short term
protocol that measures effects 48 hours following an acute insult while
the SOD1 mouse is a 126 day chronic treatment model. It is possible that
lower doses are protective, for example, if administered chronically over
a long time period.

[0148] Subjects. Animal models of inherited neurological diseases have
significantly advanced our understanding of the molecular pathogenesis
and hold great promise for developing potential therapeutic strategies
for translation to patients. The development of transgenic mice
expressing G93A human SOD1 as a mouse model for the human disease has
been regarded as a major breakthrough for development of ALS
therapeutics. G93A SOD1 transgenic mice develop progressive hind limb
weakness, muscle wasting, and neuropathological sequelae similar to those
observed in patients with both sporadic and familial ALS. The spinal cord
of the G93A SOD1 mouse shows progressive reactive astrogliosis, marked
neuronal atrophy, neuronal loss, and the presence of prominent
ubiquinated inclusion bodies by 90 days of age. In addition, motor
performance deteriorates as the disease progresses. The G93A SOD1 mouse
model has played a prominent role in studying disease progression and
especially for testing potential therapeutic agents, the latter in part
because these animals have a shortened life span of approximately 126
days. In the present study, G93A SOD1 mice and littermate controls are
bred from existing colonies at the Bedford VA Medical Hospital. The male
G93A SOD1 mice are mated with B6SJL females and the offspring are
genotyped by PCR using tail DNA. The number of SOD1 transgenes are
assessed by PCR to ensure that transgene copy number remains constant.
Mice are housed in micro-isolator cages in complete barrier facilities,
and all studies are performed in these facilities. A 12 hour light-dark
cycle is maintained and animals are given free access to food and water.
Control and transgenic mice of the same age (±2 days) and from the
same `f generation will be selected from multiple litters to form
experimental cohorts (n=20 per group). Standardized criteria for age and
parentage are used for placing individual mice into experimental
groups/cohorts. Wild type mice are used for initial toxicity,
tolerability, and pharmacokinetic studies and ALS mice are used for
one-month tolerability studies.

[0149] Tolerability, Dosing, and Pharmacokinetics. The tolerable dose
range and LD50 for butaclamol was determined in wild type mice by
increasing the dose b.i.d. one-fold each injection. The route of
administration was via i.p. administration, which was previously reported
to produce robust effects in behavioral pharmacology studies, and
starting dose range was guided by these prior studies (Lippmann et al.,
Life Sciences 16, 213, (1975); Humber et al., Mol. Pharmacol. 11, 833,
(1975); Voith and Herr, Psychopharmacologia 42, 11, (1975)). One goal was
to select a range of doses for the efficacy study starting ten fold below
the maximum tolerated dose. Initial pharmacokinetic (pK) studies were
conducted by giving animals a single dose, sacrificing them after 30 min,
1 h, 2 h, 4 h, 6 h, and 12 h, and dissecting brains and spinal cords and
determining drug concentration in the target tissue. Drug steady-state
level was determined in animals that had been dosed for 1 week prior to
sacrifice. The range of dosing levels of 0.01, 0.1, and 1 mg/kg once a
day were administered throughout the lives of the G93A mice.

[0150] Behavioral pharmacology. Behavioral testing for the transgenic G93A
SOD1 mice were performed during the light phase of the diurnal cycle
since these mice are sufficiently active during that time. Measurements
were made for 30 minutes after 10 minutes of acclimation to the box
(Opto-Varimex Unit, Columbus Instruments, Columbus, Ohio, USA). Counts of
horizontal and vertical motion activity were monitored and quantitative
analysis of locomotor activity (resting and ambulatory times), were
assessed. The open field box was cleaned before testing each mouse. Each
30 minutes of testing was analyzed as three periods of 10 minute
intervals to study the influence of novelty and measured behavior. Mice
were coded and investigators were blinded to the genotype and analysis.
Testing started on week 4 and performed every other week until the mice
could no longer participate.

[0151] Efficacy studies. Efficacy was measured using endpoints that
clearly indicate neuroprotective function. These include amelioration of
degenerative changes in the spinal cord, improved motor function, and
prolonged survival. Some mice cohorts were sacrificed at a predetermined
time point (120 days) for neuropathological examination, while others
were sacrificed at end stage disease using criteria for euthanasia. The
latter cohorts were followed temporally for behavioral analyses as well
as survival.

[0152] Survival. Mice were observed three times daily (morning, noon, and
late afternoon) throughout the experiment. Mice were euthanized when
disease progression was sufficiently severe that they were unable to
initiate movement and right themselves after gentle prodding for 30
seconds.

[0153] Body weights. Mice were weighed twice a week at the same time each
day. Weight loss is a sensitive measure of disease progression in
transgenic G93A SOD1 mice and of toxicity in transgenic and wild type
mice.

[0154] Motor/behavioral. Quantitative methods of testing motor function
are used including Rotarod and analysis of open field behavior. Decline
of motor function is a sensitive measure of disease onset and
progression. Behavioral testing for the transgenic G93A SOD1 mice were
performed during the light phase of the diurnal cycle since these mice
are sufficiently active during that time. Measurements were made for 30
minutes after 10 minutes of acclimation to the box (Opto-Varimex Unit,
Columbus Instruments, Columbus, Ohio, USA). Counts of horizontal and
vertical motion activity were monitored and quantitative analysis of
locomotor activity (resting and ambulatory times), were assessed. The
open field box was cleaned before testing each mouse. Each 30 minutes of
testing was analyzed as three periods of 10 minute intervals to study the
influence of novelty and measured behavior. Mice were coded and
investigators were blinded to the genotype and analysis. Testing started
on week 4 and performed every other week until the mice could no longer
participate.

[0155] Neuropathology. Selected cohorts (n=10) of treated and untreated
G93A SOD1 mice were euthanized at 120 days for isolation and analysis of
spinal cord tissue. For this purpose, mice are deeply anesthetized and
perfused transcardially with 4% buffered paraformaldehyde at the desired
time point. These studies were performed in a blinded manner, to avoid
bias in interpretation of the results. Brains were weighed, serially
sectioned at 50 pm and stained for quantitative morphology (cresyl
violet) to determine gross atrophy and identify ventral neuron loss and
astrogliosis. Remaining tissue samples/sections were stored for future
use. Stereology was used to quantify gross ventral horn atrophy, neuronal
atrophy, and neuronal loss. Remaining tissue samples/sections are stored
for prospective mechanistic analyses as necessary.

[0156] Analysis. Data sets were generated and analyzed for each clinical
and neuropathological measure. Effects on behavior and neuropathology
were compared in treatment and control groups. Dose-dependent effects
were assessed in each treatment group using multiple two-sided ANOVA
tests. Multiple comparisons in the same subject groups were dealt with
post hoc. Kaplan-Meier analysis was used for survival and behavioral
function.

[0157] Neuronal quantitation. Serial lumbar spinal cord tissue sections
(n=20) from L3-L5 spinal cord segments were used for gross spinal cord
areas and neuronal analysis. Gross areas of the spinal cord sections were
quantified from each experimental cohort using NIH Image. From the same
sections, the ventral horn was delineated by a line from the central
canal laterally and circumscribing the belly of gray matter. Absolute
neuronal counts of Nissl-positive neurons were performed in the ventral
horns in the lumbar spinal cord. Only those neurons with nuclei were
counted. All counts were performed with the experimenter (JM) blinded to
treatment conditions. Counts were performed using Image J (NIH) and
manually verified and the data represent the average neuronal number from
the sections used.

[0158] Interpretation. Compound efficacy is evaluated using behavioral and
neuropathological endpoints. Results for the test compound are compared
with results from compounds with established efficacy and neuroprotective
action in the G93A SOD1 mouse model. These experiments directly test
whether butaclamol provides therapeutic benefit and, if so, the magnitude
of the benefit. Along the way, useful information about solubility,
administration, and toxicity are also obtained. Treatment regimens that
show efficacy with respect to behavioral and neuropathological outcome
measures in the G93A mice at doses not predicted to produce
extrapyramidal effects would suggest that butaclamol is a potential
clinical lead with the probability for delaying onset or slowing the
progression of ALS in humans.

Results:

[0159] Behavioral results of Open-Field analysis showed marked significant
differences in out come measures between wild type littermate mice and
untreated G93A mutant mice (FIG. 9). In comparison to wild type mice,
there was significant increase in hyperactivity in the untreated G93A
mice in distance traveled, ambulatory counts, and ambulatory time
starting at 6 weeks through 12-13 weeks. After the 12-13 week time point,
there was a significant reduction in distance traveled, ambulatory
counts, and ambulatory time in G93A mice, in comparison to the wild type
littermate control mice. Resting time was the mirror antithesis of motor
movement measures, with a significant reduction from 6 weeks through
12-13 weeks, with increased resting time after 12-13 weeks. In contrast,
treated G93A mice showed motor performance changes in butaclamol-treated
mice related to dose administration.

[0160] Three different doses (low, medium, and high) of butaclamol were
tested. Parameters evaluated include (1) distance traveled; (2) resting
time; (3) ambulatory count; and (4) ambulatory time. These results are
presented in FIG. 9. Overall survival was also assessed (see FIG. 10),
and neuropathological analyses were performed (see FIGS. 11 and 12).

[0161] As can be seen with reference to FIG. 9, the low butaclamol dose
(0.01 mg/kg) in G93A mice paralleled the untreated G93A mice for distance
traveled, resting time, ambulatory count, and ambulatory time over the 6
week to 12-13 week time period. These findings are consistent with a
conclusion that the low dose butaclamol treatment had no significant
effect over that time period. After 12-13 weeks, mice treated with the
low butaclamol dose may have shown some improvement (i.e., behavior
trending toward wild type) at least with respect to resting time.

[0162] On the other hand, FIG. 9 demonstrates that G93A mice treated with
the medium dose of butaclamol showed behaviors not significantly
different from wild type mice in the period between 6 weeks and 12-13
weeks. Thus, the medium dose butaclamol treatment appeared to correct
these deficits otherwise observed in G93A mice in this time period.
Beyond the 12-13 week time point, behavior of G93A mice receiving
medium-dose butaclamol diverged from wild type mice, trending toward
untreated mice. Specifically, ambulatory distance, ambulatory counts, and
ambulatory time decreased, and resting time increased. However, at least
some of the parameters (e.g., resting time specifically) remained
significantly different from untreated mice.

[0164] No significant extrapyramidal effects were observed with any of the
treated mice. A climbing assay was used to test for extrapyramidal
effects.

[0165] Butaclamol treatment with low and medium doses extended survival in
the G93A ALS mice in a dose dependent manner (FIG. 10). The medium dose
extended survival by 12.7% as compared with untreated G93A mice. Average
life span was 125.1±3.8 days for untreated G93A mice; 129.3±4.1
days for low dose treated G93A mice; 141.0±5.3 days for medium dose
treated G93A mice; and 123.7±6.2 days for high dose treated G93A mice.

[0167] Re-purposing a known clinical agent for a new indication is by far
the most rapid path to bring a new therapy to patients. However, this
approach often has significant limitations. First of all, many compounds
are optimized for high selectivity/specificity for their initially
intended target and robust off target effects are uncommon. Second, many
compounds have a pharmacologically limited route of administration (e.g.,
topical) that may be desirable for their initial approved use but may bar
the application for another disease. This is a particular challenge for a
neurodegenerative disease therapeutic, since the vast majority of drugs
do not penetrate the blood brain barrier. The demonstration that
butaclamol has activity as a potential therapeutic for ALS is
particularly provident. Butaclamol is an orally active, blood brain
barrier penetrating compound that was designed for long term use.
Therefore butacamol has already surmounted many barriers for use as an
ALS therapeutic.

EQUIVALENTS

[0168] Having described some illustrative embodiments of the invention, it
should be apparent to those skilled in the art that the foregoing is
merely illustrative and not limiting, having been presented by way of
example only. Numerous modifications and other illustrative embodiments
are within the scope of one of ordinary skill in the art and are
contemplated as falling within the scope of the invention. In particular,
although many of the examples presented herein involve specific
combinations of method acts or system elements, it should be understood
that those acts and those elements may be combined in other ways to
accomplish the same objectives. Acts, elements, and features discussed
only in connection with one embodiment are not intended to be excluded
from a similar role in other embodiments. Further, for the one or more
means-plus-function limitations recited in the following claims, the
means are not intended to be limited to the means disclosed herein for
performing the recited function, but are intended to cover in scope any
means, known now or later developed, for performing the recited function.

[0169] Use of ordinal terms such as "first", "second", "third", etc., in
the claims to modify a claim element does not by itself connote any
priority, precedence, or order of one claim element over another or the
temporal order in which acts of a method are performed, but are used
merely as labels to distinguish one claim element having a certain name
from another element having a same name (but for use of the ordinal term)
to distinguish the claim elements. Similarly, use of a), b), etc., or i),
ii), etc. does not by itself connote any priority, precedence, or order
of steps in the claims. Similarly, the use of these terms in the
specification does not by itself connote any required priority,
precedence, or order.

[0170] The foregoing written specification is considered to be sufficient
to enable one skilled in the art to practice the invention. The present
invention is not to be limited in scope by examples provided, since the
examples are intended as a single illustration of one aspect of the
invention and other functionally equivalent embodiments are within the
scope of the invention. Various modifications of the invention in
addition to those shown and described herein will become apparent to
those skilled in the art from the foregoing description and fall within
the scope of the appended claims. The advantages and objects of the
invention are not necessarily encompassed by each embodiment of the
invention.

Patent applications by Donald R. Kirsch, Bedford, MA US

Patent applications by Radhia Benmohamed, Tewksbury, MA US

Patent applications by CAMBRIA PHARMACEUTICALS, INC.

Patent applications in class Pentacyclo ring system having the six-membered hetero ring as one of the cyclos

Patent applications in all subclasses Pentacyclo ring system having the six-membered hetero ring as one of the cyclos